The past few years have heralded a sharp increase in the availability of new approaches to gene therapy, and the number of diseases that appear potentially amenable to treatment using so-called "therapeutic genes". Generally, a therapeutic gene is a gene that corrects or compensates for an underlying protein deficit or, alternately, that is capable of down-regulating a particular gene, or counteracting the negative effects of its encoded product, in a given disease state or syndrome. Moreover, a therapeutic gene can be a gene that mediates cell killing, for instance, in the gene therapy of cancer. A successful therapeutic outcome using gene therapy hinges on the appropriate expression of the therapeutic gene, and potentially its long-term persistence in the host. Whereas expression can typically be effectively controlled using various cloning strategies that are well known to those skilled in the art, persistence (i.e., the long-term expression of the gene in the host cell) has proven more elusive.
As a general approach toward obtaining prolonged gene expression, researchers employ as a vehicle for transfer of the therapeutic gene a vector which demonstrates longevity in the host. Stability is maximized with use of a vector that integrates into the genome of the host, allowing for simultaneous integration of the therapeutic gene carried by the vector. Along these lines, retrovirus, which as part of its life cycle integrates into the host genome, has proven a useful tool. However, the use of retrovirus is not without its attendant problems. For instance, stable integration of a retroviral vector is confined to target cells that are actively synthesizing DNA; its carrying capacity is limited due to the relatively small size of the vector; the vector exhibits a lack of tissue tropism; and such a vector is rapidly inactivated by antibodies when given systemically. As a consequence of these and other shortcomings which accompany the use of a retrovirus, many researchers have turned to adenovirus (Ad) as an alternate vector for gene therapy (see, e.g., Rosenfeld et al., Science, 252, 431-434 (1991); Rosenfeld et al., Cell, 68, 143-155 (1992)).
Ads are nonenveloped, regular icosahedrons, 65-80 nanometers in diameter, that consist of an external capsid and an internal core (Ginsberg (ed.), The Adenoviruses, Plenum Press, NY (1984)). The Ad core contains a linear, double-stranded DNA molecule. Two human serotypes, namely Ad2 and Ad5, have been studied intensively and have provided the bulk of available information about Ads. This information, as well as the various useful properties of Ad (among other things, replication-deficient recombinant viruses are easily made and produced in large quantities using complementing cell lines, Ad is capable of infecting almost all cell types including terminally differentiated or non-proliferative cells, and no malignancies have been associated with Ad infection) have enabled the exploitation of Ad as an efficient gene delivery vector both in vivo and in vitro (see, e.g., Rosenfeld et al. (1991), supra; Rosenfeld et al. (1992), supra; Engelhardt et al., Hum. Gene Ther., 4, 79-769 (1993); Crystal et al., Nat. Genet., 8, 42-51 (1994); Lemarchand et al., Circ. Res., 72, 1132-1138 (1993); Guzman et al., Circ. Res., 73, 1202-1207 (1993); Bajocchi et al., Nat. Genet., 3, 229-234 (1993); Mastrangeli et al., J. Clin. Invest, 91, 225-234 (1993)).
Despite these advantages of Ad vectors, the long-term expression of an administered therapeutic gene has not satisfactorily been obtained using Ad as a gene transfer vehicle. First generation Ad vectors deleted the essential E1 region of the virus (Rosenfeld et al. (1992), supra; Boucher et al., Hum. Gene Ther., 5, 615-39 (1994)). Trans gene product from these viral vectors is detected for approximately two weeks in the rodent model system before returning to background levels. In comparison, in immune-suppressed mice, the length of the period during which gene expression can be detected following infection is substantially increased, as is the level of expression (Yang et al., J. Virol., 69, 2004-15 (1995); Yang et al., Proc. Natl. Acad. Sci., 10, 4407-11 (1994)). These data suggest that immune surveillance is responsible for the relatively poor performance of the first generation Ad vectors. Moreover, the inability of Ad to be maintained in a cell integrated into the host cell genome means that cells which express the therapeutic gene encoded by the vector are ultimately lost from the cell.
Accordingly, many researchers working with Ad have sought alternative means of stabilizing recombinant vectors such that long-lived gene expression can be obtained. One such means that has proven effective in other systems is that of maintaining the integrity of the transferred gene in the host in the form of an episome. An episome is an extrachromosomal genetic element that replicates independently of the host cell genome. To this end, the capability of Epstein-Barr virus (EBV) to form an episome in its latent state has been usurped by researchers as a means to generate episomes from various double-stranded DNA templates.
EBV is a human lymphotropic herpes virus which causes infectious mononucleosis and is associated with at least two human cancers (Reisman et al., Molec. Cell. Biol., 5, 1822-1832 (1985)). EBV contains two origins of replication, oriLyt and oriP. During latency, EBV exists as a closed circular double-stranded DNA molecule which employs oriP in its replication, and which is maintained at a copy number ranging from 10 to 200 per cell (Young et al., Gene, 62, 171-185 (1988)). Plasmids that harbor oriP can be maintained in cells that also express the nuclear antigen EBNA-1 (see, e.g., Yates et al., Nature, 313, 812-815 (1985); Jalanko et al., Biochemica et Biophysica Acta, 949, 206-212 (1988); Kioussis et al., EMBO J., 6, 355-361 (1987); Jalanko et al., Arch. Virol., 103, 157-166 (1988); Sugden et al., J. Virol. 63, 2644-2649 (1989)). In the presence of EBNA-1, oriP permits plasmid replication in a variety of mammalian cells that EBV is incapable of infecting in culture (Reisman et al., supra; Yates et al., Proc. Natl. Acad Sci., 81, 3806-3810 (1984)). The oriP origin contains two cis-acting elements that are required for its activity (reviewed in Middleton et al., Advances in Virus Research, 40, 19-55 (1991)). The elements are separated by 1,000 base pairs (bp), and both are composed of multiple degenerate copies of a 30 bp segment. The first element, termed the family of repeats, or FR, contains 20 tandem 30 bp repeats. The second element is comprised of a 114 bp segment that contains a 65 bp dyad symmetry element, or DS.
Mutagenesis studies reveal that the two regions of oriP function vis-a-vis each other in an orientation- and distance-independent manner (Middleton et al., supra). Deletion of the intervening spacer region, or addition to this region of more than 1,000 bp, does not affect the function of oriP. The FR element is known to function as an enhancer, but its role in replication remains unclear. Moreover, the 20 tandem repeats comprising FR can be replaced by multiple copies of DS. As few as eight of the 20 tandem repeats found in the FR enhancer are sufficient in short term assays for both enhancer activity and plasmid replication.
EBNA-1 binds to the 30 bp repeats present in both elements of oriP. The EBNA-1 protein is required for the initiation of DNA replication near DS which occurs once per cycle during the S phase of the EBV cell cycle (Middleton et, al., supra). A glycine-alanine repetitive sequence which comprises approximately 1/3 of the protein and a small region in the carboxyl terminus of the protein is dispensable for plasmid replication (Yates et al. (1985), supra; Lupton et al., Mol. Cell. Biol., 5, 2533-2542 (1985)). However, this sequence prevents the immune system from detecting EBNA-1 and, consequently, would appear necessary for persistence of EBV and EBV-derived vectors (Levitskaya et al., Nature, 375, 685-688 (1995)).
The stability of episomes obtained using EBV is potentially limiting for long-term persistence and expression of a therapeutic gene inasmuch as there is at least some possibility of an error in partitioning newly synthesized episomes between daughter cells, and in view of the pervasive negative selection pressure with respect to extrachromosomal genetic elements. Integration of the episome into a nondeleterious locus in the genome, i.e., a safe haven for the transferred gene, would obviate this potential lack of stability. Along these lines, adeno-associated virus (AAV) demonstrates a unique ability to integrate with high frequency into human chromosome 19q13.3-qter (Kotin et al., Human Gene Therapy, 5, 793-801 (1994)). This ability of AAV to integrate into a defined and benign genomic site eliminates the risk of insertional mutagenesis due to inadvertent gene activation or inactivation that accompanies random insertion of DNA (Shelling et al., Gene Therapy, 165-169 (1994)).
In terms of its general features, AAV is a human parvovirus that can be propagated either as an integrated provirus, or by lytic infection (Muzyczka, Current Topics in Microbiol. and Immunol., 158, 97-129 (1992)), and which has been employed as a vector for eukaryotic cells (see,. e.g., U.S. Pat. Nos. 4,797,368 and 5,173,414; Tratschin et al., Mol. Cell. Biol., 4, 2072-2081 (1984); GenBank Database Accession Number J01901 or AA2C). The lytic phase of AAV infection requires the expression of the Ad early gene products E1a, E1b, E2a, E4, and VA RNA (Kotin et al., supra). Latent infections are established by infection of AAV in the absence of helper virus. Under these circumstances, AAV efficiently integrates into the cellular genome, and is maintained in that state unless challenged with Ad.
Only two components of AAV are required for locus-directed integration of a foreign gene into the human genome: the Rep proteins, and the AAV inverted terminal repeats (ITRs). The four Rep proteins are each encoded by the same gene, and are generated by alternative splicing of nascent mRNAs (Kyostio et al., J. Virol., 68, 2947-2957 (1994)). The two larger Rep proteins, Rep78 and Rep68, bind the AAV ITRs and act as ATP-dependent, sequence-specific endonucleases with helicase activity to unwind the region of the ITRs during AAV DNA replication (Holscher et al., J. Virol., 68, 7169-7177 (1994)). The smaller Rep proteins, Rep52 and Rep40, appear essential for the accumulation of single-stranded progeny genomes used in packaging the virus.
The AAV ITRs are located at each end of the genome. When the viral ITRs are single-stranded, they form T-shaped hairpin structures (Snyder et al., J. Virol., 67, 6096-6104 (1993)). The origin of replication, packaging, integration and excision signals are also found located within the region of the viral ITRs. Binding of Rep proteins to the AAV ITRs is consistent with the essential role of this region in ITR-dependent replication and locus-directed integration. In the absence of Rep proteins, replication does not occur, and integration into the genome appears to be a random event (Kotin et al., supra). The ITRs can function either in their native state or as cloned into a plasmid as double-stranded DNA. With appropriate application of Rep proteins (e.g., by providing Rep proteins through coding sequences located in trans), foreign sequences incorporated between the ITRs can be excised from a plasmid, replicated and integrated into the host cell genome.
The implementation of the EBV strategy to generate an episome, or the AAV strategy to stabilize an episome or sequences carried by the episome through incorporation into a host cell genome, requires tight regulation of the relevant regulatory protein in each system (e.g., EBNA-1 for EBV, and Rep proteins for AAV). One approach that has been employed by researchers for achieving tight regulation of gene expression is to control expression of a gene or genetic sequence via a site-directed recombination event. Two well studied systems that allow site-specific recombination and have been used in a variety of applications are the phage P1 Cre/Lox system and the yeast Flp/Frt system.
The Cre and Flp proteins belong to the .lambda. integrase family of DNA recombinases (reviewed in Kilby et al., TIG, 9, 413-421 (1993); Landy, Current Opinion in Genetics and Development, 3, 699-707 (1993); Argos et al., EMBO J., 5, 433-440 (1986)). The Cre and Flp recombinases show striking similarities, both in terms of the types of reactions they carry out and in the structure of their target sites and mechanism of recombination (see, e.g., Jayaram, TIBS, 19, 78-82 (1994); Lee et al., J. Biolog. Chem., 270, 4042-4052 (1995); Whang et al., Molec. Cell. Biolog., 14, 7492-7498 (1994); Lee et al., EMBO J., 13, 5346-5354 (1994); Abremski et al., J. Mol. Biol., 192, 17-26 (1986); Adams et al., J. Mol. Biol., 226, 661-673 (1992)). For instance, the recombination event is independent of replication and exogenous energy sources such as ATP, and functions on both supercoiled and linear DNA templates.
The Cre and Flp recombinases exert their effects by promoting recombination between two of their target recombination sites, Lox and Frt, respectively. Both target sites are comprised of inverted palindromes separated by an asymmetric sequence (see, e.g., Mack et al., Nucleic Acids Research, 20, 4451-4455 (1992); Hoess et al., Nucleic Acids Research, 14, 2287-2300 (1986); Kilby et al., supra). The asymmetry provides directionality to the recombination event. Namely, recombination between target sites arranged in parallel (i.e., so-called "direct repeats") on the same linear DNA molecule results in excision of the intervening DNA sequence as a circular molecule (Kilby et al., supra). Recombination between direct repeats on a circular DNA molecule excises the intervening DNA and generates two circular molecules. In comparison, recombination between antiparallel sites (i.e., sites which are in opposite orientation, or so-called "inverted repeats") on a linear or circular DNA molecule results in inversion of the internal sequence. Even though recombinase action can result in reciprocal exchange of regions distal to the target site when targets are present on separate linear molecules, intramolecular recombination is favored over intermolecular recombination.
Both the Cre/Lox and Flp/Frt recombination systems have been used for a wide array of purposes. For instance, site-specific integration into plant, insect, bacterial, yeast and mammalian chromosomes has been reported (see, e.g., Sauer et al., Proc. Natl. Acad. Sci., 85, 5166-5170 (1988); Fukushige et al., Proc. Natl. Acad. Sci., 89, 7905-7907 (1992); Baubonis et al., Nucleic Acids Research, 21, 2025-2029 (1993); Hasan et al., Gene, 150, 51-56 (1994); Golic et al., Cell, 59, 499-509 (1989); Sauer, Mol. Cell. Biolog., 7, 2087-2096 (1987); Sauer et al., Methods: Companion to Methods in Enzymol., 4, 143-149 (1992); Sauer et al., The New Biologist, 2, 441-449 (1990); Sauer et al., Nucleic Acids Res., 17, 147-161 (1989); Qin et al., Proc. Natl. Acad. Sci., 91, 1706-1710 (1994); Orban et al., Proc. Natl. Acad. Sci., 89, 6861-6865 (1992)). Eukaryotic viral vectors have been assembled, and inserted DNA has been recovered, using these systems (see, e.g., Sauer et al., Proc. Natl. Acad. Sci., 84, 9108-9112 (1987); Gage et al., J. Virol., 66, 5509-5515 (1992); Holt et al., Gene, 133, 95-97 (1993); Peakman et al., Nucleic Acids Res., 20, 495-500 (1992)). Specific deletions of chromosomal sequences and rearrangements have also been engineered, and excision of foreign DNA as a plasmid from .lambda. vectors is presently possible (see, e.g., Barinaga, Science, 265, 27-28 (1994); Rossant et al., Nature Medicine, 1, 592-594 (1995); Sauer, Methods in Enzymol., 225, 890-900 (1993); Sauer et al., Gene, 70, 331-341 (1988); Brunelli et al., Yeast, 9, 1309-1318 (1993); InVitrogen (San Diego, Calif.) 1995 Catalog, 35; Clontech (Palo Alto, Calif.) 1995/1996 Catalog, 187-188). Cloning schemes have been generated so that recombination either reconstitutes or inactivates a functional transcription unit by either deletion or inversion of sequences between recombination sites (see, e.g., Odell et al., Plant Physiol., 106, 447-458 (1994); Gu et al., Cell, 73, 1155-1164 (1993); Lakso et al., Proc. Natl. Acad. Sci., 89, 6232-6236 (1992); Fiering et al., Proc. Natl. Acad. Sci., 90, 8469-8473 (1973); O'Gorman et al., Science, 251, 1351-55 (1991); Jung et al., Science, 259, 984-987 (1993)). Similarly, positive and negative strategies for selecting or screening recombinants have been developed (see, e.g., Sauer et al., J. Mol. Biol., 223, 911-928 (1992)). The genes encoding the Cre or Flp recombinases have been provided in trans under the control of either constitutive, inducible or developmentally-regulated promoters, or purified recombinase has been introduced (see, e.g., Baubonis et al., supra; Dang et al., Develop. Genet., 13, 367-375 (1992); Chou et al., Genetics, 131, 643-653 (1992); Morris et al., Nucleic Acids Res., 19, 5895-5900 (1991)). The use of the recombinant systems or components thereof in transgenic mice, plants and insects among others reveals that hosts express the recombinase genes with no apparent deleterious effects, thus confirming that the proteins are generally well-tolerated (see, e.g., Orbin et al., Proc. Natl. Acad. Sci., 89, 6861-6865 (1992)).
More recently, researchers have employed replication-deficient Ad vectors containing the phage P1 gene for Cre as a means of studying intracellular Cre-mediated recombination (Anton et al., J. Virol., 69, 4600-4606 (1995)). In these experiments, the Cre-expressing Ad vector was supplied to cells along with another Ad vector, in which the coding sequence of a reporter gene was separated from any promoter by an extraneous spacer sequence flanked by parallel Lox sites. Cre-mediated recombination resulted in excision of the spacer sequence, and a turning on of the formerly silent reporter gene. This approach would appear to allow for only the positive modulation of gene expression, and not stable gene expression, inasmuch as the gene switched on by the recombination event will be expressed only as long as the replication-deficient Ad vector is maintained in the host cell. Moreover, there is a possibility of the reverse recombination reaction simultaneously switching off the reporter gene and imposing an upper limit on the expression level to be obtained, due to continuing production of Cre within the host cell (Anton et al., supra, page 4605).
Accordingly, there remains a need for expression systems in which the potential of Ad and other gene therapy vectors can be more fully realized. The present invention seeks to provide such expression systems. In particular, it is an object of the present invention to provide methods for site-specific recombination in a cell, and vectors which can be employed in such methods, which allow prolonged gene expression as well as modulation of gene expression, and which overcome some of the aforementioned problems inherent in prior gene expression systems. These and other objects and advantages of the present invention, and additional inventive features, will be apparent from the description of the invention provided herein.